The present application is directed to droplet ejection, and more specifically, to the generation and transmission of droplets to analytical instruments such as mass spectrometers.
Mass spectrometers are an analytical tool concerned with the separation of molecular (and atomic) species according to their mass and charge. More particularly, it is an analytical tool used for measuring the molecular weight (MW) of a sample. For large samples such as biomolecules (e.g., analyte molecules), molecular weights can be measured within an accuracy of 0.01% of the total molecular weight of the sample, i.e., within 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. More commonly, the accuracy is about 0.05% or 20 Da with a good measurement and multiple charge states present. This is sufficient to allow minor mass changes to be detected, e.g., the substitution of one amino acid for another. Often, however, there are substitutions for amino acids that do not significantly alter the mass (i.e., isoleucine and leucine have the same mass), these substitutions may pose problems for any mass spectrometer. For small organic molecules, the molecular weight can be measured to within an accuracy usually sufficient to confirm the molecular formula of a compound. To achieve such measurements, a mass spectrometer of sufficient resolution will of course be required.
Mass spectrometers are particularly useful for analyzing the products of chemical reactions, since they can identify specific products by their mass signature. However, large complexes, such as protein-ligand interactions have been difficult to detect using conventional mass spectrometry methods. This is in part due to the fact the introduction and charging of the sample is a relatively violent process, which tends to break up larger molecules into fragments. Fortunately, new sample introduction techniques have been developed, such as electrospray ionization (ESI), which are much less violent, making it possible to now detect protein ligand complexes directly in mass spectrometers. Such mass spectrometers are ESI-quadrupole mass spectrometers, one in particular being ESI time-of-flight of mass spectrometers (ESI-TOF mass spectrometers).
There are numerous types of mass spectrometers, in addition to those known as time-of-flight. However, the basic components are similar. As shown in FIG. 1, mass spectrometer 10 can be divided into three fundamental parts. First, an ion source 12 ionizes the molecules of interest, then a mass analyzer 14 differentiates the ions according to their mass-to-charge ratio, and finally a detector 16 measures an ion beam current. Each of these elements can take many forms and are combined to produce a wide variety of mass spectrometers with specialized characteristics.
The analyzer and detector of the mass spectrometer, and often the ionization source, are maintained under high vacuum 17 to provide the ions a path to travel from one end of the instrument to the other without hindrance from other molecules. The entire operation of the mass spectrometer, and often the sample introduction process, is under a system controller 18.
As may be apparent from the foregoing, an important aspect of mass spectrometry is sample introduction into the instrument 10. The sample inlet 19 is the interface between the sample and the mass spectrometer. One approach is to place a sample on a probe which is then inserted, usually through a vacuum lock, into the ionization region of the mass spectrometer 10. The sample can then be heated to facilitate thermal desorption or undergo any number of high-energy desorption processes used to achieve vaporization and ionization.
Capillary infusion is often used because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a mass spectrometer with other separation techniques, including, but not limited to, gas chromatography, liquid chromatography (LC), high pressure liquid chromatography (HPLC) or capillary electrophoresis (CE).
Up until twenty years ago, practical techniques for interfacing liquid chromatography (LC) with available ionization techniques were not available. A major problem prohibiting this interface was getting the sample stream from the liquid chromatographic process into the mass spectrometer without losing vacuum while also ionizing the sample. However, new ionization techniques, such as the previously mentioned electrospray ionization, allow liquid chromatography/mass spectrometry processes to be routinely performed. One configuration of a liquid chromatography/mass spectrometry design 20 is shown in FIG. 2. More particularly, a pump 22 moves a sample liquid to chromatographic separation columns 24 wherein the liquid sample is separated into a series of components. The liquid sample is then sent to a capillary spray nozzle or needle 26, and electrospray 28 ionization processes 28 are performed 28. The ionized sample is then introduced to mass analyzer 30, and detected by detector 32 for generation of output signals 34.
As previously noted, the ionization source in this example is an electrospray ionization system. A more detailed view of such a system is shown as electrospray configuration 40 of FIG. 3. In this design, a liquid sample 42, which may be provided from the liquid chromatography process of FIG. 2 moves through a metal capillary or needle 44, which has an open end with a sharply pointed tip, such as the end of a syringe. This tip is attached to a voltage supply 45 of between approximately 1.5 kv to 4 kv, depending upon the implementation. The end of the tip faces a counterelectrode plate or cylinder electrode 46 held at a voltage lower than the tip voltage to generate a voltage gradient. As the voltage in the liquid is applied, the liquid becomes charged, generating a force sufficient to expel the liquid from the capillary tip. As the liquid sample 42 pushes from the tip, a shape described as a Taylor cone is developed. At the very end of the cone, the droplets push away from one another into a fine spray 48, at times called a plume. It is to be appreciated that a capillary LC process uses pump pressure to expel the fluid.
Depending on the electric field used, the charges may be positive or negative. The droplets may contain both solvent molecules, as well as analyte (sample) molecules and may be less than 10 micrometers across. The droplets move across the electric field, and with the assistance of a flow of N2 gas, provided by gas inlet 50, neutral solvent molecules are evaporated from the droplets. As the droplets become smaller, and the total charge on the droplets stays the same, so the concentration of charge increases. Eventually, at what is known as the Rayleigh limit, Coulombic repulsion overcomes the droplet surface tension and the droplet explodes. The Coulombic explosion forms a series of smaller, lower-charged droplets. This process of shrinking followed by explosion is repeated, until eventually the analyte (sample) molecule is stripped of solvent molecules, and is left as a charged ion. These ions are then moved through capillary 52, which is in a differently pumped region 54. A skimmer 56 may be provided to further refine the sample.
A major instrumental challenge with electrospray ionization is the interface between the ion source (which may be at atmospheric pressure) and the mass spectrometer (at a high vacuum, one example value may be about 10xe2x88x926 torr; it is to be appreciated however that this value is instrument dependent). This problem is addressed by the use of a pinhole aperture 58 around 10-100 micrometers. In this design, the created ions are drifted (with the help of the electric field) towards the aperture. The emitted drops are then, as previously noted, analyzed in the analysis section and then provided to a detector for generation of output signals.
It is to be appreciated that in addition to the described electrospray technique, other ionization designs are known, such a nanospray technology. This process is similar to electrospray technology, but uses a smaller capillary, and requires less sample solution, i.e., it is a low flow rate version of electrospray ionization.
Thus, as shown above, use of electrospray ionization and nanospray ionization, provides increased uses of mass spectrometry. Particularly, these techniques permit for the integration of separation functions, such as liquid chromatography type technology with the mass spectrometry.
Unfortunately, electrospray and/or nanospray sample introduction remains relatively slow, since it relies on the plumbing of fluids through small capillary tubes prior to droplet generation with a high electric field. For operations which would benefit from high throughput, such as drug screening and others, it is desirable to have sample introduction techniques which are substantially faster.
Another drawback of the techniques described is the potential for contamination. Specifically, since the sample is required to pass through substantial amounts of tubing and come into contact with a number of distinct surfaces, the potential for contamination of the sample exists.
Further, in areas such as combinatorial chemistry and others, the sample fluids are contained within wells of a well-plate. Often it is desired to review the reaction of byproducts resulting from the combination of the fluids in the wells. Presently it is necessary to process the fluids through the capillary tubing required by existing liquid chromatography/mass spectrometry configurations. Thus a drawback exists in that it is necessary to remove the fluids from the well of the well-plate prior to providing the sample fluid to the mass spectrometer. One example of providing a sample from a well-plate to a mass spectrometer is described in U.S. Patent Application 2002/0109084 A1, entitled Acoustic Sample Introduction For Mass Spectrometric Analysis, published Aug. 15, 2002, filed February 14, 2001, hereby incorporated in its entirety by reference.
Provided is a system for delivering a liquid sample to an inlet of an analytical instrument. Included is an acoustic ejector driven by a drive system that generates drive signals provided to the ejector. The drive signals are generated with a pulse width sufficient to eject at least a portion of the liquid sample. A reservoir provided for holding the liquid sample is in operational arrangement with the acoustic ejector. A liquid sample voltage source is located within the reservoir, and the liquid sample voltage source is designed to provide a charge to the liquid sample. An analytical instrument voltage source is in operational arrangement with the analytical instrument and is designed to provide a voltage bias between the reservoir and the analytical instrument.
In another aspect of the invention, provided is a method for delivering a liquid sample to an inlet of an analytical instrument. The method includes generating an acoustic drive signal, with a pulse width sufficient to eject at least a portion of the liquid sample below a short pulse limit. The drive signal is delivered to an acoustic ejector whereby the acoustic ejector generates an acoustic wave. The acoustic wave is directed into a reservoir holding the liquid sample, wherein the acoustic wave is focused substantially at a surface of the liquid sample. This design results in the emitting of at least a portion of the liquid sample.